Method for detection of l523s expression in biological samples

ABSTRACT

The present invention discloses methods, for differentiating between normal or reactive cells and malignant cells biological samples comprising: exposing the biological sample to an antibody to L523S protein and detecting the presence of the antibody bound to L523S protein within the malignant cells. In embodiments, such methods may further comprise: exposing the biological sample to an antibody to a second protein that is a marker of cell lineage and detecting the presence of the antibody to the second protein within cells corresponding to a particular cell lineage and/or may comprise: exposing the biological sample to an antibody to a third protein which is a marker of cells that are normal or reactive and detecting the presence of the antibody to the third protein within cells that are normal or reactive.

TECHNICAL FIELD

The present invention relates to the use of an antibody to L523S in thedifferentiation of malignant cells from benign, reactive or normal cellsin biological samples.

BACKGROUND OF INVENTION

Some of the primary challenges faced by pathologists in cancer diagnosesrelate to distinguishing between different types of malignancies anddifferentiating between benign and malignant cell proliferations.

One example of such type of challenge is in the diagnosis of lungneuroendocrine tumors, the spectrum of which includes typical carcinoid,atypical carcinoid, large cell neuroendocrine carcinoma (LCNEC) andsmall cell carcinoma (SCLC). This spectrum of lung neuroendocrine tumorsprovides a range of phenotypically distinct entities with differentbiological behaviors. Although various types of lung neuroendocrinetumors share common neuroendocrine features, they differ greatly intheir cytogenetic and molecular characteristics, highlighting afundamental molecular divergence among these tumors. Importantly, theyexhibit different degrees of aggressiveness, with typical carcinoid andSCLC representing the two extremes of malignancy.

In the second instance, benign conditions, including inflammatoryconditions, infectious processes and benign cell proliferations can bedifficult to distinguish from malignancy. As one example, the positiveidentification of malignant cells in lung pleural tissue biopsies and inpleural effusions can be challenging, particularly when both normal andreactive mesothelial cells are present. Pathologists generally rely oncharacteristic histologic features to aid in differentiating betweenbenign and malignant conditions, however benign/reactive cells can oftenshare these histologic features with malignant cells. Furthermore, whenevaluating specimens derived from biologic fluids and needle biopsies,it can be difficult to obtain specimens with sufficient morphologicdetail for clinical diagnosis. Another example is in differentiatingbenign melanocytic nevi from malignant melanoma. Benign nevi, especiallyin those with atypical features can be difficult to distinguish frommalignant melanoma.

Another approach, which has lately been widely used in cancerdiagnostics, is molecular diagnostics. There have been describednumerous molecular markers of cancer cells, such as particularabnormalities in genetic material associated with particular forms ofcancer, abnormalities on the transcription level, specific profiles ofthe expressed proteins.

L523S protein is one of the promising molecular markers of cancer cellswhich can be used for the molecular cancer diagnostics. The protein isalso known as K homology domain containing protein overexpressed incancer (KOC), which has been described by Mueller-Pillasch et al.(Oncogene 1997, 14(22), pp. 2729-2733) and by Nielsen et al. (Mol. CellBiol. 1999, 19(2), pp. 1262-1270). Nielsen et al. (Mol. Cell Biol. 1999,19(2), pp. 1262-1270) have determined that L523S (KOC) is identical toIMP3, a member of the insulin-like growth factor II (IGF-II)mRNA-binding protein (IMP) family. The L523S protein is a 580 amino acidoncofetal RNA binding protein that has four K homology domains and thatregulates insulin-like growth factor II (IGF-II) transcripts duringembryogenesis. L523S is later re-expressed in a proportion of cancercells from various types of tumors, including pancreatic and lungadenocarcinoma, and it may function in promoting tumor cellproliferation by enhancing IGF-II protein expression.

Evidence has previously been presented (for instance, in US PatentApplication Publications 2003/0236209 A1, 2003/0138438 A1 and2002/0147143 A1) indicating that L523S may also be present in colonadenocarcinomas, prostate adenocarcinomas, CML, AML, Burkitt's Lymphoma,brain tumors, retinoblastomas, ovarian tumors, teratocarcinomas, uterusmyosarcomas, germ cell tumors as well as pancreatic and cervical tumorcell lines. Since it was first identified in pancreatic carcinomas byGress, et al. (Oncogene 1996, 13, pp. 1819-1830), the marker L523S hasbeen detected in non-small cell carcinomas of the lung as well as otherhuman malignancies. In particular, immunohistochemical studies haveindicated L523S expression is a marker of increasingly aggressivebiologic potential among pancreatic ductal lesions (Istvanic et al.,Mod. Pathol. 2005, 18(S1); pp. 298A-299A). Strong and diffuseimmunohistochemical expression of this marker is highly sensitive forthe identification of invasive carcinoma and can be found in bothseverely dysplastic lesions as well as carcinomas of the pancreas. Inaddition, Jiang et al. (Lancet Oncol. 2006, 7, pp. 556-564) have shownthat L523S is expressed in renal cell carcinoma.

Various DNA encoding sequences for and amino acid sequences of L523Shave been taught in U.S. Pat. Nos. 6,969,518; 6,960,570; 6,696,247;6,531,315; 6,518,256; 6,482,597 and in United States Patent ApplicationPublications 2003/0236209 A1; 2003/0138438 A1; 2002/0147143 A1;2002/0115130 A1 and 2002/0052329 A1.

Using rtPCR and microarray analyses, it has been found (Wang et al., BrJ Cancer 2003, 88(6), pp. 887-894) that L523S mRNA expression is minimalin many normal tissues. Importantly, however, L523S is widely expressedin many malignancies. Istvanic et al. (Mod. Pathol. 2005, 18(S1); pp.298A-299A) have found that a subset of carcinomas from variousorgans—lung, pancreas, esophagus, stomach, colorectum, cervix andbladder—express the L523S protein. The restricted and relatively lowlevel expression of L523S in a subset of normal tissue types compared toits presence in many different forms of cancer make it a potentialmarker of malignancy.

Despite of accumulated evidence indicating that expression of the L523Sprotein is associated with metastatic potential of cancer cells, fulldiagnostic and prognostic potential of estimating the expression ofL523S protein in cells for molecular diagnostics of cancer and, thus,the value of L523S protein a cancer biomarker have not beeninvestigated.

SUMMARY OF INVENTION

The present invention relates to use of L523S protein as a cancerbiomarker in methods for diagnosis, prognosis, monitoring of cancer andtreatment decisions, which based on immunodetection of the protein inmalignant cells of biological samples and, thus, differentiating betweennormal or reactive cells and malignant cells of said biological samples.According to the invention differentiating between cells of the samplesis possible because the expression of L523S can be detected only inmalignant cells but not in normal or reactive cells present in thesamples.

In general, a cancer may be detected in a patient by immunohistochemicaltest methods or flow cytometry based on the presence of one or moredistinctive tumor proteins in a biological sample (for example, blood,sera, sputum, pleural fluid, peritoneal fluid, urine and/or tumor tissuebiopsies) obtained from the patient. In other words, such proteins maybe used as markers to indicate the presence or absence of a cancer suchas lung cancer. In addition, such proteins may be useful for thedetection of other cancers.

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect polypeptide markers in asample. In general, one commonly used detection assay format willcomprise the steps of (a) contacting a biological sample obtained from apatient with a binding agent such as an antibody or antibodies; (b)detecting in the sample a level of polypeptide that binds to the bindingagent, possibly by detecting a label associated with the antibody orantibodies. The level of polypeptide bound to the binding agent iscommonly concerned with the presence or absence of a disease.

The present invention employs anti-L523S antibodies to distinguishbetween benign and malignant cells in various biological samples. Suchbiological sample could be from either humans or animals and could behistological samples, e.g. tissue and/or cell specimens, including celllines, tissues, cell preparations, blood, bodily fluids, otherbiological fluids, bone marrow, cytology specimens, blood smears,thin-layer preparations, imprints, cytospins, cell block sections,micro-arrays, and, frequently, biological samples, such as biopsyspecimens, disposed on microscope slides.

In addition, the invention uses an antibody to a marker of cell lineagetogether with anti-L523S to aid in the clinical evaluation bydetermining the tissue of origin of cells, which can be important forprognosis and for treatment decisions. Furthermore, the inventionemploys antibodies to proteins whose expression is restricted tobenign/reactive cells in the panel to confirm the diagnosis of benignprocesses.

Thus, a first aspect the invention relates to a method fordifferentiating between normal or reactive cells and malignant cells inbiological samples characterized by:

-   -   exposing the biological sample to an antibody to L523S protein;        and    -   detecting the presence of the antibody bound to L523S protein        within the malignant cells.

Another aspect of the invention relates to a method for differentiatingbetween normal or reactive cells and malignant cells in a tissue samplecharacterized by:

-   -   exposing a first portion of the tissue sample to an antibody to        L523S protein; and    -   detecting the presence of the antibody bound to L523S protein        within the malignant cells.

Still, in another aspect, the invention relates to a method fordifferentiating between normal or reactive cells and malignant cells ina biological sample comprising a cell monolayer or cell pellet derivedfrom a biological fluid characterized by:

-   -   exposing a first portion of the biological sample to an antibody        to L523S protein; and    -   detecting the presence of the antibody bound to L523S protein        within the malignant cells.

All abovementioned aspects of the invention may comprise detection ofother biomarkers specific for reactive and/or normal cells which may bepresent in a sample selected for the immunochemical detection of theL523S protein. As mentioned above, such detection may be advantageous tothe diagnosis of benign processes or treatment decisions. In embodimentswhich comprise such detection, the abovementioned methods may compriseexposing the biological sample to an antibody to a second protein thatis a marker of cell lineage and detecting the presence of the antibodyto the second protein within cells corresponding to a particular celllineage. In embodiments, such methods may further comprise exposing thebiological sample to an antibody to a third protein which is a marker ofcells that are normal or reactive and detecting the presence of theantibody to the third protein within cells that are normal or reactive.The second protein may be calretinin and the third protein may bedesmin. The detecting steps may comprise immunohistochemical staining orflow cytometry.

Still, a further aspect of the invention relates to a panel ofbiomarkers which may be used for the purpose of differentiating betweennormal or reactive cells and malignant cells in a biological sample.Further, the invention relates to a kit for the differentiation betweennormal or reactive cells and malignant cells in a biological sample,wherein the kit comprises a panel of antibodies to the proteins of thedescribed panel, wherein at least one of the antibodies is an anti-L523Santibody, at least one another antibody is an antibody a second proteinwhich a marker of a particular cell lineage, and at least one anotherantibody is an antibody to a third protein which is a marker of cellsthat are normal or reactive. In particular such kit comprises ananti-L523S antibody, an antibody to calretinin and antibody to desmin.

DESCRIPTION OF DRAWINGS

FIG. 1A is a flow chart that schematically illustrates a first methodfor distinguishing malignant tissue or cells in accordance with theinvention.

FIG. 1B is a flow chart that schematically illustrates a second methodfor distinguishing malignant tissue or cells in accordance with theinvention.

FIG. 2A shows a photomicrograph of normal lung pleura immunostained withanti-Calretinin demonstrating normal mesothelial cells positivelystained in a line on the edge of the tissue.

FIG. 2B shows a photomicrograph of a serial section of the normal lungpleura of FIG. 1A immunostained with anti-L523S demonstrating a lack ofimmunoreactivity of anti-L523S with normal mesothelial cells present.

FIG. 3A shows a photomicrograph of reactive mesothelial cells within anormal lung pleural biopsy immunostained with anti-Calretinin,demonstrating positively stained reactive mesothelial cells.

FIG. 3B shows a photomicrograph of a serial section of the lung pleuralbiopsy of FIG. 3A immunostained with anti-desmin demonstratingpositively stained reactive mesothelial cells.

FIG. 3C shows a photomicrograph of a serial section of the lung pleuralbiopsy of FIG. 3A immunostained with anti-L523S demonstrating a lack ofimmunostaining of anti-L523S with reactive mesothelial cells.

FIG. 4A is a photomicrograph of a lung pleural biopsy with mesotheliomaimmunostained with anti-L523S.

FIG. 4B is a photomicrograph of a lung pleural biopsy with melanomaimmunostained with anti-L523S.

FIG. 4C is a photomicrograph of a lung pleural biopsy with lungadenocarcinoma immunostained with anti-L523S.

FIG. 4D is a photomicrograph of a lung pleural biopsy with lung spindlecell carcinoma immunostained with anti-L523S.

FIG. 4E is a photomicrograph of a lung pleural biopsy with lung squamouscell carcinoma immunostained with anti-L523S.

FIG. 5A is a photomicrograph of a lung pleural biopsy containingmesothelioma immunostained with anti-L523S, demonstrating cytoplasmicstaining within the cancer cells.

FIG. 5B is a photomicrograph of a serial section of the lung pleuralbiopsy of FIG. 4A immunostained with anti-desmin showing positivestaining of only muscle elements of the tissue.

DISCLOSURE OF INVENTION

In order to overcome the aforementioned challenges in the art, novelmethods for detection of L523S expression in biological samples areherein disclosed.

Before the present invention is described, it is to be understood thatthis invention is not limited to the particular embodiments described,as such methods, devices, and formulations may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. It must be noted that as used herein and in theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise, and includesreference to equivalent steps and methods known to those skilled in theart.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the specificmethods and/or materials in connection with which the publications arecited.

1. Methods for Differentiating Between Normal or Reactive Cells andMalignant Cells

As it is mentioned above a first aspect of the invention relates to amethod for differentiating between normal or reactive cells andmalignant cells in biological samples characterized by:

-   -   exposing the biological sample to an antibody to L523S protein;        and    -   detecting the presence of the antibody bound to L523S protein        within the malignant cells.

By the term “differentiating” in the present context is meant toidentify a difference between cells present in a biological sample, inparticular in a sample which comprise a plurality of cells correspondingto different cell lineages, such as for example cells of a biopsysample. By “cell lineage” is meant a pedigree of cells related throughmitotic division. Some embodiments of the invention particularly relate,but not limited, to mesothelial cell lineages, which may bemorphologically characterized by the presence of flattened epithelialcells of mesenchymal origin that line the serous cavities of the body.

By “biological sample” is meant a sample comprising biological material,in particular a sample of comprising cells, e.g. a sample comprisingcells derived from a cell culture, for example cell pellet or cellsuspension, sample of body tissue or body fluid, biopsy sample. By“tissue sample” is meant a collection of similar cells obtained from atissue of a subject or patient, preferably containing nucleated cellswith chromosomal material. The four main human tissues are (1)epithelium; (2) the connective tissues, including blood vessels, boneand cartilage; (3) muscle tissue; and (4) nerve tissue. The source ofthe tissue sample may be solid tissue as from a fresh, frozen and/orpreserved organ or tissue sample or biopsy or aspirate; blood or anyblood constituents; bodily fluids such as cerebral spinal fluid,amniotic fluid, lung pleural effusion, peritoneal fluid, or interstitialfluid; cells from any time in gestation or development of the subject.The tissue sample of the invention may also be primary or cultured cellsor cell lines. The tissue sample may contain compounds which are notnaturally intermixed with the tissue in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

In one embodiment of the invention, the biological sample is a biopsysample, in another embodiment of the invention the biological sample isa sample of any tissue identified above, in another embodiment thebiological sample may be a sample of bodily fluids, in still anotherembodiment the biological sample may be a sample of cell specimens,including cell lines, tissues, cell preparations, blood, otherbiological fluids, bone marrow, cytology specimens, blood smears,thin-layer preparations, imprints, cytospins, cell block sections,micro-arrays, and, frequently, biological samples, such as biopsyspecimens, disposed on microscope slides cultured cells or a samplederived from cultured cells, such as cell pellet or culture mediumcollected from cultured cells. Particular non-limited examples of theinvention include a biopsy of pigmented skin lesion, monolayer or pelletor cell block section derived from a biological fluid, wherein the fluidis lung pleural effusion.

The above mentioned and below described methods include according to theinvention assaying biological samples for the presence of particularcells, such as malignant, normal and/or reactive cells by employingantibodies to specific biomarkers of said cells.

The term “antibody” refers to an immunoglobulin molecule or a fragmentthereof having the ability to specifically bind to a particular antigen.The antibody may be an anti-marker protein antibody specific for theprotein used in the assay. Thus, the antibody may be capable ofspecifically binding the protein marker as the antigen. Antibodies andmethods for their manufacture are well known in the art of immunology.The antibody may be produced, for example, by hybridoma cell lines, inwhich case the antibody will be capable of specific binding to a singlespecific site on the antigen termed “epitope”, and the antibodycharacterized by this feature is termed “monoclonal antibody”, byimmunization to elicit a polyclonal antibody response, which means thatan antibody capable of recognizing multiple sites, epitopes, on theantigen is produced, or by recombinant host cells that have beentransformed with a recombinant DNA expression vector that encodes theantibody. Antibodies include but are not limited to immunoglobulinmolecules of any isotype (IgA, IgG, IgE, IgD, IgM), and active fragmentsincluding Fab, Fab′, F(ab′)₂, Facb, Fv, ScFv, Fd, V_(H) and V_(L).Examples of antibodies include but are not limited to single chainantibodies, chimeric antibodies, mutants, fusion proteins, humanizedantibodies and any other modified configuration of an immunoglobulinmolecule that comprises an antigen recognition site of the requiredspecificity. The invention relates to any type of antibody molecule or afragment thereof which is suitable for use in the methods disclosedherein. The invention relates to antibody fragments which remain thecapability of the corresponding antibody to specifically bind to theantigen. The invention relates to both primary and secondary antibodies.The “primary antibody” is an antibody which is specific for a particularantigen. By “secondary antibody” is meant an antibody which is capableof specifically recognizing an immunoglobulin molecule. Thus, thesecondary antibody may be defined as an antibody against the primaryantibody.

As it has already been mentioned an antibody of the invention may be anyantibody or a fragment thereof, however, in some embodiments, amonoclonal antibody may be preferred. Monoclonal antibodies contain onlyone single species of antibody combining site, or paratope, capable ofimmunoreacting with a particular epitope. However, a monoclonal antibodymay comprise more than one specific antibody combining site, suchantibodies being polyspecific, e.g. bispecific. Suitably the presentmonoclonal antibodies are monospecific and comprise a single paratopespecific for the marker to be detected.

The preparation of antibodies including antibody fragments and othermodified forms is described, for example, in “Immunochemistry inPractice,” Johnstone and Thorpe, Eds., Blackwell Science, Cambridge,Mass., 1996; “Antibody Engineering,” 2^(nd) edition, C. Borrebaeck, Ed.,Oxford University Press, New York, 1995; “Immunoassay”, E. P. Diamandisand T. K. Christopoulos, Eds., Academic Press, Inc., San Diego, 1996;“Handbook of Experimental Immunology,” Herzenberg et al., Eds, BlackwellScience, Cambridge, Mass., 1996; and “Current Protocols in MolecularBiology” F. M. Ausubel et al., Eds., Greene Pub. Associates and WileyInterscience, 1987, the disclosures of which are incorporated herein. Inparticular, the preparation of monoclonal antibodies, which is wellknown in the art, was first disclosed by Koehler and Milstein, Nature,256: 496-497, 1975.

Assaying the samples include a step of detecting the antibody bound tothe corresponding biomarker. Accordingly, an antibody of the inventionmay be detectably labeled. Numerous labels are available which can begenerally grouped into the following-categories:

-   -   (a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and 131I. The        antibody can be labeled with the radioisotope using the        techniques described in Current Protocols in Immunology, Volumes        1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y.,        Pubs. (1991) for example and radioactivity can be measured using        scintillation counting.    -   (b) Colloidal gold particles.    -   (c) Fluorescent labels including, but are not limited to, rare        earth chelates (europium chelates), Texas Red, rhodamine,        fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin,        phycocyanin, or commercially available fluorophores such        SPECTRUM ORANGE® and SPECTRUM GREEN® and/or derivatives of any        one or more of the above. The fluorescent labels can be        conjugated to the antibody using the techniques disclosed in        Current Protocols in Immunology, supra, for example.        Fluorescence can be quantified using a fluorimeter.    -   (d) Various enzyme-substrate labels are available and U.S. Pat.        No. 4,275,149 provides a review of some of these. The enzyme        generally catalyzes a chemical alteration of the chromogenic        substrate that can be measured using various techniques. For        example, the enzyme may catalyze a color change in a substrate,        which can be measured spectrophotometrically. Alternatively, the        enzyme may alter the fluorescence or chemiluminescence of the        substrate. Techniques for quantifying a change in fluorescence        are described above. The chemiluminescent substrate becomes        electronically excited by a chemical reaction and may then emit        light which can be measured (using a chemiluminometer, for        example) or donates energy to a fluorescent acceptor. Examples        of enzymatic labels include luciferases (e.g., firefly        luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),        luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase,        urease, peroxidase such as horseradish peroxidase (HRPO),        alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,        saccharide oxidases (e.g., glucose oxidase, galactose oxidase,        and glucose-6-phosphate dehydrogenase), heterocyclic oxidases        (such as uricase and xanthine oxidase), lactoperoxidase,        microperoxidase, and the like. Techniques for conjugating        enzymes to antibodies are described in O'Sullivan et al.,        Methods for the Preparation of Enzyme-Antibody Conjugates for        use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone        & H. Van Vunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

-   -   (i) Horseradish peroxidase (HRP) with hydrogen peroxidase as a        substrate, wherein the hydrogen peroxidase oxidizes a dye        precursor [e.g., orthophenylene diamine (OPD) or        3,3′,5,5′-tetramethyl benzidine hydrochloride (TMB)];    -   (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate        as chromogenic substrate; and    -   (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate        (e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate        (e.g., 4-methylumbelliferyl-β-D-galactosidase).

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label may be indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thefour broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody may be conjugated with a small hapten and one ofthe different types of labels mentioned above is conjugated with ananti-hapten antibody. Thus, indirect conjugation of the label with theantibody can be achieved. In some embodiments of the invention one ormore secondary antibody molecules may be conjugated with alabel-conjugated polymer backbone. Thus, amplification of the signal maybe achieved.

Indirectly and directly labeled secondary antibodies are alsocommercially available. For example, one example of commerciallyavailable label-conjugated polymer backbone carrying secondary antibodymolecules reagent is EnVision™ reagent (DAKO). A secondary antibodycarrying a label aimed for a particular type of detection may beobtained from numerous manufacturers.

According to the invention, detecting the antibody bound to a markerprotein, such as L523S or other protein of the invention, may comprisedetecting an immunohistochemical stain. Thus, a method fordifferentiating between normal or reactive cells and malignant cells inbiological samples comprising a step of immunohistochemical detection(IHC) of a marker protein is one of the embodiments of the invention.

Two general methods of IHC are available at present: direct and indirectassays. According to the first assay, binding of antibody to the targetprotein is determined directly. This direct assay uses a labeledreagent, such as a fluorescent tag or an enzyme-labeled primaryantibody, which can be visualized without further antibody interaction.Such detection is preferred embodiment of the invention. In a typicalindirect assay, unconjugated primary antibody binds to theprotein-antigen and then a labeled secondary antibody binds to theprimary antibody. Where the secondary antibody is conjugated to anenzymatic label, a chromagenic or fluorogenic substrate is added toprovide visualization of the antigen. Signal amplification occursbecause several secondary antibodies may react with different epitopeson the primary antibody. Using labeled secondary antibodies is anotherpreferred embodiment of the invention. Further amplification of thesignal maybe achieved when a detection method comprises using alabel-conjugated polymer backbone that carries a number of secondaryantibody molecules. Such method is another embodiment of the invention.Particular embodiments of these detection methods relate to anenzyme-labeled secondary antibodies and enzyme-conjugated polymerbackbone that carries secondary antibody molecules. The examples ofenzymes may be selected but not limited to commonly used horse radishPeroxidase (HRP) or alkaline phosphatase (AP).

The sample preparation procedures, treatment of the tissue section priorto, during or following IHC may be desired. For example, epitoperetrieval methods, such as heating the tissue sample in citrate buffermay be carried out [see, e.g., Leong et al. Appl. Immunohistochem.4(3):201 (1996)].

Following an optional blocking step, the tissue section is exposed toprimary antibody for a sufficient period of time and under suitableconditions such that the primary antibody binds to the target proteinantigen in the tissue sample. Appropriate conditions for achieving thiscan be determined by routine experimentation.

A signal for the bound antibody may be amplified by using well-known inthe art methods and reagents available from commercial manufacturers.

Specimens thus prepared may be mounted and coverslipped. Slideevaluation is then determined, e.g. using a microscope.

The extent of binding of antibody to the sample is determined by usingany one of the detectable labels discussed above. Some times may bepreferred the label which is an enzymatic label (e.g. horse radishperoxidase) which catalyzes a chemical alteration of the chromogenicsubstrate vial as 3,3′-diaminobenzidine chromogen. Preferably theenzymatic label is conjugated to antibody which binds specifically tothe primary antibody (e.g. the primary antibody is rabbit polyclonalantibody and secondary antibody is goat anti-rabbit antibody).

The antibodies and labeled antibodies according to the invention may beused in a variety of immunoimaging or immunoassay procedures to detectthe presence of cells expressing the L523S protein or cells expressingother marker protein in a biological sample. This aims to detect acancer in a patient or monitor the status of such cancer in a patientalready diagnosed to have it. When used to monitor the status of acancer a quantitative immunoassay procedure may be used. Such monitoringassays are carried out periodically and the results compared todetermine whether the patients tumor burden has increased or decreased.Common assay techniques that may be used include direct and indirectassays. Direct assays involve incubating a tissue sample or cells fromthe patient with a labeled antibody. If the sample comprise L523Sprotein bearing cells such as includes lung cancer or cells chosen fromthe group consisting of melanoma, mesothelioma, lung adenocarcinoma,spindle cell carcinoma, squamous cell carcinoma, colonic adenocarcinoma,ovarian carcinoma, gastric carcinoma, pancreatic carcinoma,asctrocytoma, Hodgkin's lymphoma, lymphoma, malignant fibroushistocytoma, Ewing's sarcoma and carcinoid, the labeled antibody of theinvention will bind to those cells. After washing the tissue or cells toremove unbound labeled antibody, the tissue sample is read for thepresence of labeled immune complexes.

Labeled by antibodies cells in fluid samples may be identified by flowcytometry which is commonly used for this purpose in the art.

All the above disclosed embodiments relate to all methods of theinvention aimed for the differentiating between normal or reactive cellsand malignant cells in biological samples. In particular, these methodsinclude methods comprising detecting a single biomarker of malignantcells, the L523S protein, such as:

1. a method for differentiating between normal or reactive cells andmalignant cells in biological samples characterized by:

-   -   exposing the biological sample to an antibody to L523S protein;        and    -   detecting the presence of the antibody bound to L523S protein        within the malignant cells;

2. a method for differentiating between normal or reactive cells andmalignant cells in a tissue sample characterized by:

-   -   exposing a first portion of the tissue sample to an antibody to        L523S protein; and    -   detecting the presence of the antibody bound to L523S protein        within the malignant cells.

3. a method for differentiating between normal or reactive cells andmalignant cells in a biological sample comprising a cell monolayer,pellet or cell block section derived from a biological fluidcharacterized by:

-   -   exposing a first portion of the biological sample to an antibody        to L523S protein; and    -   detecting the presence of the antibody bound to L523S protein        within the malignant cells;

In some embodiments the methods of the inventions may include one ormore additional steps comprising detecting the presence of one or moreother protein markers specific for other particular types of cellspresent in the analyzed samples. In particular, the invention relates tomethods for differentiating between normal or reactive cells andmalignant cells in biological samples which comprise the steps ofexposing the biological sample or a portion thereof to an antibody toL523S protein and detecting the presence of the antibody bound to L523Sprotein within the malignant cells, and one or more additional steps,such as the steps of:

-   -   exposing the biological sample, or a portion thereof, to an        antibody to a second protein that is a marker of cell lineage;    -   and detecting the presence of the antibody to the second protein        within cells corresponding to a particular cell lineage;

and/or the steps of

-   -   exposing the biological sample, or a portion thereof, to an        antibody to a third protein which is a marker of cells that are        normal or reactive; and    -   detecting the presence of the antibody to the third protein        within cells that are normal or reactive.

In particular, the invention relates to one or more other proteinmarkers selected from protein markers of normal or reactive, or proteinsthat are markers of particular cell lineages. Specifically, theinvention relates to calretinin as a marker of a particular celllineage, e.g. a mesothelial cell lineage, and desmin as a marker ofnormal or reactive cells, such as benign cells. However, the inventionis not limited to these particular protein markers. A selection of suchprotein marker will in most embodiments depend on particular type ofbiological sample to be analyzed, depending on cellular phenotypespresent in the sample.

By “portion” of biological sample, in particular “portion” of tissuesample is meant a “section” or “part” of a solid or fluid sample or cellsample by which is meant a single part or piece of said sample, e.g. athin slice of tissue or cells cut from a tissue sample. It is understoodthat multiple sections of tissue samples may be taken and subjected toanalysis according to the present invention, provided that it isunderstood that the present invention comprises a method whereby thesame section of biological sample may be analyzed at both morphologicaland molecular levels, or is analyzed with respect to one or moremolecular markers, e.g. L523S, desmin and/or calretinin. In someembodiments different portions of the same solid tissue sample orbiological fluid sample may be analyzed for the presence of differentmolecular markers described herein.

All methods of the invention may comprise one or more further sub-steps,such as detection sub-steps steps which may improve or enhance thesignal, for example the sub-steps of:

-   -   further exposing the biological sample, or a portion thereof, to        a labeled secondary antibody that reacts with the antibody bound        to L523S protein, or with the antibody bound to a protein which        is a marker of cells that are normal or reactive, or with the        antibody bound to a protein which is a marker a particular cell        lineage; and    -   detecting the presence of the labeled secondary antibody;

or sub-steps of:

-   -   further exposing the biological sample, or a portion thereof, to        a labeled reagent comprising an enzyme-conjugated polymer        backbone that carries secondary antibody molecules that react        with the antibody bound to L523S protein, or with the antibody        bound to a protein which is a marker of cells that are normal or        reactive, or with the antibody bound to a protein which is a        marker a particular cell lineage; and    -   detecting the presence of the labeled reagent.

According to the invention the above described methods are useful fordifferentiating between normal or reactive cells and malignant cells inbiological samples wherein the type of malignant cells is chosen fromthe group consisting of melanoma, mesothelioma, lung adenocarcinoma,spindle cell carcinoma, squamous cell carcinoma, colonic adenocarcinoma,ovarian carcinoma, gastric carcinoma, pancreatic carcinoma,asctrocytoma, Hodgkin's lymphoma, lymphoma, malignant fibroushistocytoma, Ewing's sarcoma and carcinoid

2. Protein Panels and Kits

The present invention also discloses a panel of protein markers whichare useful for differentiating between normal or reactive cells andmalignant cells in biological samples, said panel comprising a marker ofmalignant cells, a marker of normal or reactive cells and a marker ofcells of a particular cell lineage. Particularly, the invention relatesto a panel consisting of L523S protein as a marker of malignant cells,desmin as a marker or normal or reactive cells, and calretinin as amarker of a particular cell lineage. Use of this panel of proteinmarkers can be aid in the clinical evaluation by determining the tissueof origin of cells, which can be important for prognosis and fortreatment decisions. The markers are identified in the correspondingcells by antibodies to these markers. Accordingly, in a further aspect,the invention relates to a kit which comprises a panel of antibodiescapable of recognizing the panel of biomarkers identified above. Thepanel of antibodies according to the invention comprises at least oneantibody reagent to a protein marker of malignant cells, at leastantibody reagent to a protein marker of a particular cell lineage and/orat least one antibody reagent to a protein marker of normal or reactivecells. In some embodiments the panel may comprise two or more differentantibody reagent to any of the members of the abovementioned panel ofbiomarkers, wherein at least one of said antibodies is capable ofrecognizing and specifically binding to a marker of malignant cells andat least one another antibody is capable of recognizing and specificallybinding to a marker of a particular cell lineage. Preferably, themalignant cell marker is L523S protein, the normal or reactive cellmarker is desmin, and the cell lineage marker is calretinin.

A kit comprising a panel of antibodies of the invention may compriseboth primary antibodies and secondary antibodies. In such embodiment thesecondary antibodies may contain a detectable label. In some embodimentsthe kit may contains several different secondary antibodies which maycomprise different labels. This may be aid to simultaneous detection ofthe panel biomarker described above. In some embodiments the kit maycomprise only primary antibodies. In some embodiments the primaryantibodies of such kit may be labeled. Preferably, the primaryantibodies comprised by the kit are monoclonal antibodies.

The may also comprise other reagents necessary for the detection of thelabel.

A particular kit of the invention may consists of an antibody to L523Sprotein, an antibody to desmin and an antibody to a marker of aparticular cell lineage and further comprises one or more non-primaryantibody reagents. Another particular kit may consists of an antibody toL523S protein, an antibody to calretinin and an antibody to a marker ofcells with are normal or reactive, and further comprises one or morenon-primary antibody reagents. In still another particular embodimentthe kit may comprise an antibody to L523S protein, an antibody to desminand an antibody to calretinin, and further comprises one or morenon-primary antibody reagents. The wording “comprise” in the presentcontext means that the kit consists of the indicated antibodies andother reagents, which are not primary antibodies to other proteinmarkers. However, in some embodiments, the kits of the invention mayfurther comprise additional primary antibody reagents to other cellmarkers of the discussed above cellular types, i.e. malignant cellmarkers, normal or reactive cell markers and cell lineage markers

A kit comprising the panel of antibodies to the markers described aboveis successfully used for analysis of cells of biopsy samples ofpigmented skin and diagnosis of melanoma. Therefore, a kit comprisingsuch panel of antibodies may be in one embodiment aimed for thedetection of melanoma. In other embodiments the kit may be used for thedetection of mesothelioma, lung adenocarcinoma, spindle cell carcinoma,squamous cell carcinoma, colonic adenocarcinoma, ovarian carcinoma,gastric carcinoma, pancreatic carcinoma, asctrocytoma, Hodgkin'slymphoma, lymphoma, malignant fibrous histocytoma, Ewing's sarcoma orcarcinoid

EXAMPLES

MATERIALS: In a first set of experiments, formalin-fixed paraffinembedded (FFPE) human lung pleural tissue biopsies were obtained andimmunohistochemically examined. These samples were derived fromtwenty-eight cases consisting of nine normal lung pleura and lung pleuracontaining the following malignancies: mesothelioma (thirteen cases),melanoma (a single case), adenocarcinoma (three cases), spindle cellcarcinoma (a single case) and squamous cell carcinoma (a single case).Two cases of normal pericardial tissue containing an intact mesotheliallayer were also obtained. Additionally, FFPE biopsies of malignanttumors of colonic adenocarcinoma, ovarian carcinoma, gastric carcinoma,pancreatic carcinoma, astrocytoma, Hodgkin's lymphoma, lymphoma,malignant fibrous histocytoma, Ewing's sarcoma and carcinoid wereobtained and examined. Some of the tissue samples were provided by theCooperative Human Tissue Network, which is funded by the National CancerInstitute.

In another set of experiments, a total of forty-six surgically resectedlung neuroendocrine tumor specimens from forty-six patients wereimmunohistochemically studied according to the methods of the invention.These samples were retrieved from the surgical pathology archives fromJanuary 1996 to March 2006 at University of Rochester Medical Center andfrom January 1994 to January 2005 at Washington University MedicalCenter. These lung neuroendocrine tumor specimens included sixteentypical carcinoids, six atypical carcinoids, fourteen LCENCs and tenSCLCs.

In a third set of experiments, fifty-one samples with paraffin embeddedpleural effusion cell blocks were retrieved and immunohistochemicallystudied according to methods of the invention. These samples hadpreviously been diagnosed as either benign (sixteen) or malignantpleural effusion (thirty-five, including ten malignant mesotheliomas,ten lung adenocarcinomas, seven breast adenocarcinomas, four colonicadenocarcinomas, three small cell carcinomas and a single squamous cellcarcinoma).

In a fourth set of experiments, eighty slides containing benignurothelium and various urothelial lesions were immunohistochemicallystudied. In a final, fifth, set of experiments, thirty-three surgicallyexcised or biopsied melanocytic lesions, including fourteen malignantmelanomas (comprising thirteen from skin with Breslow depth from 0.275mm to 1.3 cm and one from metastasis in soft tissue of the arm), eightdysplastic nevi, and eleven benign nevi (all of which may, collectively,be referred to as pigmented skin lesions) were immunohistochemicallystudied.

Procedures.

The tissue biopsies were sectioned by microtome at 4 um and mounted oncharged slides. The tissue samples on slides were deparaffinizedaccording to established procedures and, and, in some cases, rehydratedin graded alcohols. Generally, the samples were then washed in runningwater and Tris buffer saline (TBS) consisting of 50 mM Tris-HCl (pH7.6), 150 mM NaCl and 0.05% Tween 20. Next, the slides were pretreatedwith heat-induced epitope retrieval in Target Retrieval Solution pH 9.0or pH 6.0 (provided by Dako, Glostrup Denmark), and cooled for betweenfifteen and twenty minutes at room temperature.

Immunohistochemistry was performed using the EnVision+ System, HRP(Dako) and performed either manually (with or without humid chamber) orusing the Dako Autostainer. Endogenous peroxidase was quenched byincubating sections in 3% hydrogen peroxide for five to six minutes.Mouse monoclonal anti-human L523S (Dako) clone 69.1 was used at aconcentration of 0.5 μg/ml on tissue pretreated with a 30-40 minuteheating protocol. Mouse monoclonal anti-human desmin (Dako) clone D33was used at 4.7 μg/ml on tissue pretreated with a 20-minute heatingprotocol. Lung pleural tissue biopsies and pleural effusions were alsostained with calretinin. For these, mouse monoclonal anti-humancalretinin (Dako) clone DAK Calret 1 was used at 1.3 μg/ml on tissuepretreated with a 20-minute heating protocol. Lung pleural effusionsamples were also stained with CK 5/6. In many cases, a negative controlreagent was also used with each tissue sample to confirm the specificityof the immunostaining. The various primary antibodies were applied tothe tissue and incubated for between thirty and sixty minutes, dependingupon sample, and slides were rinsed with Tris-Buffered Saline with Tween(TBST). Bound antibody was detected by incubating with aperoxidase-conjugated Envision+ polymer detection system, which, inaddition, also carries secondary link antibody molecules (rabbit ormouse) (Dako), or goat-anti-rabbit or horse-anti-mouse IgG-biotin andstreptavidin-HRP for 30 minutes. Finally, the slides were incubated in3,3′-diaminobenzidine (DAB+) substrate/chromogen solution orAEC+Substrate-Chromogen (Dako), depending upon sample, for 10 minutes,rinsed in DI water, counterstained with hematoxylin and mounted andviewed under a light microscope.

Accordingly, modes for carrying out the invention are summarized in andmay be understood with reference to FIG. 1A and FIG. 1B, which are flowcharts that schematically illustrate first and second methods fordifferentiating between normal or reactive cells and malignant cells ina biological sample in accordance with the present invention. The firstmethod 15, schematically illustrated in FIG. 1A, comprises two steps. Inthe first step 17, the biological sample, or a portion of the biologicalsample, is exposed to an antibody to L523S protein, so as to enable theantibody to bind to this marker protein (if any). Subsequently, in thesecond step 19, the antibody that has bound to L235S within the sampleis then detected. A detected level or amount above or greater somepre-determined threshold is then taken as a positive indication ofmalignancy or of the aggressiveness or grade of the malignancy. As oneexample, both the intensity of cytoplasmic staining (0 to 3+) and thepercentage of cells stained (0-100%) may be recorded in step 19, withpositive staining being defined as 2+ or 3+ intensity (for example) in10% or more cells (for example). As another example, positive stainingmay be defined as instances in which more than 10% of cells showcytoplasmic staining of any staining intensity.

In general, the detection of the bound antibody will be achieved bydetecting some label, such as a chromogen or fluorochrome. In general,this detection will be through microscopic analysis, either manual orautomated. The biological sample will often be a tissue biopsy but may,instead, be a biological fluid, such as a sample of lung pleuraleffusion. Often, only a portion of a biological sample, rather than theentire biological sample, will be exposed to an antibody to L523Sprotein in step 17. If the sample is a tissue sample, the portion may,for instance, be a thin slice of the tissue sample as produced by amicrotome. If the sample is of a body fluid or derived or taken from abody fluid, then the sample portion may assume one of a variety ofdifferent physical configurations, such as a cell monolayer formed byswabbing or smearing the fluid onto a microscope slide (e.g., a smear)or by centrifugation of the fluid or a cell pellet formed bycentrifugation of the fluid. Although the intensity and location ofstaining of stained biological samples are frequently determinedvisually by microscopy of samples mounted on slides, flow cytometrytechniques could instead be used to detect and enumerate cells(disaggregated and derived from either from either tissue or fluidsamples) having one or more types of chemical labels specific to themarkers described herein.

The second method 25, schematically illustrated in FIG. 1B, represents amodification of the first method 15. In the first step 27 of method 25,a biological sample is exposed to an antibody to L523S protein and isalso exposed to an antibody to at least one other marker protein, so asto enable the antibody to L523S to bind to L523S in the sample (if any)and so as to enable the second antibody to bind to the at least oneother marker protein. The second marker protein may, for instance, be amarker to cell lineage, such as calretinin, which is a marker ofmesothelial cell lineage. Alternatively, the second marker protein maybe a marker of cells that are normal or reactive, such as desmin. Morethan one such “second” marker may be employed in a single set of testsor on a single sample. Subsequently, in the second step 29, the boundantibodies (if any) are detected as described previously. These multiplemarkers may be detected in an individual section or specimen (e.g., atissue section or a cell monolayer or pellet derived or prepared from abody fluid) through multi-staining of the section or specimen.Alternatively, in this method, it may be advantageous to exposedifferent portions of the sample, such as serial sections from a samplebiopsy, to the different respective antibodies. If the sample is of abody fluid or taken from a body fluid, then the different portions maycomprise different sections of one or more cell pellets or may comprisedifferent cell monolayer preparations formed by centrifugation orsmearing.

Results

Results from Studies of Lung Pleural Tissue Biopsies. In order toappreciate the practice and advantages of the present invention, thereader is referred to the accompanying FIGS. 2-5 in conjunction with thefollowing discussion, which illustrate and describe immunohistochemicalstaining results biopsies of lung pleural tissue. It is to be noted thatthe accompanying FIGS. 2-5 are only intended to be exemplary of theresults obtained by employing methods in accordance with the presentinvention and are not intended to be in any way limiting of theinvention. In the accompanying figures, staining with the DAB+substrate-chromogen solution results in a brown-colored precipitate(appearing as dark gray to black in the accompanying drawings) at theantigen sites whereas the counterstaining with hematoxylin results inthe blue-purple precipitate haematein (appearing as light to medium grayin the accompanying drawings) at cell nuclei. Cells staining positivefor Calretinin and desmin also appear as dark gray to black in theaccompanying drawings.

Although the present invention is described in terms ofimmunohistochemical staining methods that produce readily visualizedcolored precipitates within biological samples, it is to be understoodthat the invention is considered to encompass a variety of knownimmunoshistochemical techniques for visualization of antigen sites. Forinstance, the so-called “direct” method of immunohistochemical stainingcould be used, in which a single labeled antibody that binds directly tothe target antigen is detected, either optically or otherwise.Alternatively, any “indirect method” of staining may be employed,wherein an unlabeled primary antibody reacts with tissue antigen, and alabeled secondary antibody that reacts with the primary antibody isdetected. Still further, the staining method may include utilizing,instead of the simple secondary antibody, a reagent of the typedescribed in U.S. Pat. No. 5,543,332, incorporated herein by reference,in which secondary antibody molecules are carried on anenzyme-conjugated polymer backbone. The enzyme, when allowed to catalyzea substrate so as to form a colored reaction product, acts as a label ofthe reagent which can be detected via the colored reaction product. Suchreagents are marketed under the trade name EnVision by Dako of GlostrupDenmark. In either case, the label could comprise, for instance, eitherindividually or in combination, a chromogen, a fluorescent dye such asFITC, rhodamine or Texas red, an enzyme such as peroxidase, alkalinephosphatase or glucose oxidase, a chemiluminescent, material, or amolecule containing a radioactive isotope, magnetic particles, etc.

Returning, once again, to the discussion of the figures, normalmesothelial cells 2 were Identified in human lung pleural biopsies usingimmunohistochemistry by their positivity for Calretinin (FIG. 2A,dark-colored zone) and lack of L523S expression (FIG. 2B). Reactivemesothelial cells 4 positively immunostained for Calretinin (FIG. 3A,dark colored region) and desmin (FIG. 3B), but were also negative forL523S (FIG. 3C). In all, nine normal lung pleural biopsies, anti-L523Swas unreactive with normal and reactive mesothelial cells, whileanti-desmin positively immunostained both normal and reactivemesothelial cells in eight of the nine normal lung pleural biopsies.Desmin expression was found to be more intense in reactive mesothelialcells compared to normal mesothelial cells. Normal human pericardialmesothelial cells were found to be immunoreactive with anti-L523S,demonstrating that specific types of mesothelial cells can express L523Sprotein (data not shown).

Using the novel immunohistochemistry staining method described above,L523S protein expression—that is, cells 6 staining positive forL523S—was identified in malignant cells in eight of thirteen (includingtwo weak positives) lung pleural biopsies containing mesothelioma (FIG.4A); in one (of one) lung pleural biopsy containing melanoma (FIG. 4B);in three of three lung pleural biopsies containing adenocarcinoma (FIG.4C); in one (of one) lung pleural biopsy containing spindle cellcarcinoma (FIG. 4D) and in one (of one) lung pleural biopsy containingsquamous cell carcinoma (FIG. 4E). Anti-L523S also demonstrated someimmunoreactivity in non-cancer cells and in secretions such as: lungsecretions (rare), subset of normal bronchi (faint staining), germinalcenter of the lymph node and reactive fibroblasts or activatedlymphocytes (rare). In contrast, anti-desmin was unreactive withmalignant cells, though many normal muscle cells were positivelyimmunostained in the biopsies (FIG. 5B). Interestingly, cells notidentified as muscle did positively immunostain with anti-desmin in twocases of mesothelioma. These small groups of cells are likely reactivemesothelial cells. FIGS. 5A-5B demonstrate the contrasting stainingpattern seen with anti-L523S compared to anti-desmin on a lung pleuralbiopsy containing mesothelioma, where L523S immunoreactivity can be seenwithin the cytoplasm of the cancer cells (seen in FIG. 5A as cytoplasmof tumor cells 8 that are positively stained for L523S expression), anddesmin expression is present only in the muscle elements 10 of thetissue (FIG. 5B).

Results from Studies of Biopsies of Lung Neuroendocrine Tumors. In ourimmunohistochemical analyses, nine SCLCs (90%) showed a strong anddiffuse (more than 90% of tumor cells) cytoplasmic staining pattern forL523S, and only 1 case exhibited focal immunoreactivity. Nine offourteen LCNECs (64%) showed strong cytoplasmic staining detected ingreater than 90% of the tumor cells. The remaining 5 cases exhibited avariable degree of immunoreactivity, but none of the cases werecompletely negative for L523S expression. No positive staining for L523Swas detected in any of the sixteen typical carcinoids. Five of sixatypical carcinoids also showed a complete lack of immunoreactivity,with only a single atypical carcinoid exhibiting weak staining in areasexhibiting oncocytic changes.

Results from Pleural Effusion Samples. In thirty-five malignant pleuraleffusion samples positive staining for L523S in a variable degree ofintensity was observed in 28 (80%) cases, including 100% ofmesotheliomas, 100% of lung adenocarcinomas, 100% of small cell andsquamous cell carcinomas, 75% of colonic adenocarcinomas and 14% ofbreast adenocarcinomas. Sixteen cases diagnosed as reactive mesothelialcells showed positivity for calretinin and thirteen of sixteen wereCK5/6 positive. Interestingly, mesothelial cells were positive for L523Sin two of sixteen cases originally interpreted as benign. One of thesewas diagnosed as malignant mesothelioma on pleural biopsy. All malignantmesotheliomas were positive for calretinin, and eight of ten cases hadCK5/6 staining.

Results from Studies of Biopsies of Bladder Urothelial Tissue. Positivestaining was observed in the majority of lesions in the high gradegroup, including invasive urothelial carcinoma, urothelial carcinomain-situ, and high grade papillary urothelial carcinoma. No expression ofL523S was detected in low grade papillary urothelial carcinoma, and thegroup of benign to borderline lesions, including normal urothelium(fourteen samples), papillary urothelial neoplasm of low malignantpotential (three samples) and urothelial atypia of unknown significance(a single sample). Expression of L523S between low grade papillaryurothelial carcinoma and each lesion in the high grade group wasstatistically significant, as was that between benign to borderlinegroup and each lesion of the high grade group.

Results from Biopsies of Pigmented Skin Lesions. Seven of fourteen (50%)malignant melanomas, including one metastatic melanoma in soft tissue ofthe arm, showed moderate to strong positive staining for L523S, withfour cases exhibiting positivity in >90% of tumor cells and three casesshowing positivity in 10-20% of tumor cells. All four (100%) cases withBreslow depth of >1.3 mm showed moderate to strong positive staining forL523S while two of nine (22%) cases with Breslow depth 1.3 mm exhibitedmoderate to strong positive staining for L523S, and they aresignificantly different (p<0.05). No positive L523S staining wasdetected in 8 dysplastic nevi and 11 benign nevi.

Conclusions

In total, tissues from all of the following tumors have been testedaccording to the methods of the present invention and found to beimmunoreactive with anti-L523S: mesothelioma, melanoma, lungadenocarcinoma, squamous cell carcinoma, spindle cell carcinoma, colonicadenocarcinoma, ovarian carcinoma, gastric carcinoma, pancreaticcarcinoma, astrocytoma, Hodgkin's lymphoma, lymphoma, malignant fibroushistocytoma, Ewing's Sarcoma and carcinoids. As but one example,differentiating reactive mesothelial cells from malignant cells in humanlung pleural biopsies by traditional methods can be challenging.However, the novel immunohistochemistry staining method disclosed hereinprovides a new tool to aid in this differential. Anti-L523S may haveutility as a marker of malignancy in this diagnostic setting because itdoes not react with normal and reactive lung pleural mesothelial cells,but is expressed in many cancers. In this study, L523S expression wasdemonstrated in 8/13 mesotheliomas, 1/1 melanoma, 3/3 adenocarcinomas,1/1 spindle cell carcinoma and 1/1 squamous cell carcinoma found in lungpleural biopsies. While L523S was not expressed in all of the cancerouscells tested, a positive result can assist in differentiating malignantcells from benign mesothelial cells.

The use of additional antibodies such as anti-desmin and anti-calretininmay further aid in differentiating between reactive mesothelial cellsand malignant cells. Anti-desmin can confirm the presence of normal orreactive mesothelial cells on the specimen, while anti-calretinin as amesothelial cell marker can indicate that benign or malignant cells areof mesothelial origin. Further, the use of a panel of markers inimmunohistochemistry, which includes anti-L523S and anti-desmin can aidin differentiating between reactive lung pleural mesothelial cells andmalignant cells.

We also conclude that L523S is highly expressed in high grade lungneuroendocrine carcinomas, but not in low and intermediate gradecarcinoid tumors, indicating that L523S may be used as a diagnosticimmunomarker in the distinction between high grade and low/intermediategrade lung neuroendocrine tumors, particularly when the diagnosticmaterial is limited with crush artifact. We further conclude that L523Sis a sensitive and specific marker for detection of malignant cells inpleural effusion, and that it has significant utility in diagnosis ofreactive mesothelial cells, malignant mesothelioma and metastaticcarcinoma in combination with calretinin and CK5/6 staining.

We further conclude that L523S is overexpressed in high grade papillaryurothelial carcinoma and urothelial carcinoma in-situ, in comparison tobenign urothelium and borderline and low grade lesions and,consequently, immunohistochemical staining for this marker may help ingrading difficult cases of urothelial tumors. We still further concludethat L523S is expressed in malignant melanoma but not in melanocyticnevi even when dysplastic features are present, that positive detectionof this marker parallels Breslow depth of invasion and that L523S is amarker of aggressiveness in the malignant melanoma.

A novel method for immunoreaction and staining of anti-L523s on normaland malignant biological samples has been disclosed. As can be easilyunderstood from the foregoing, the basic concepts of the presentinvention may be embodied in a variety of ways. The essence of theinvention includes not only sample processing techniques but, also, thevarious systems, assemblies, and devices required or usable toaccomplish the sample processing. Various modifications and variationsof the described methods and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed is not intended to be and should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in diagnostic pathology or related fields are intended tobe within the scope of the claims. Note that any patents, patentapplications, publications, or other references mentioned in thisapplication for patent are hereby incorporated herein by reference.

1. A method of characterizing cells in a biological sample comprisingnormal or reactive and malignant cells, said method comprising: (a)obtaining a biological sample; (b) contacting the biological sample withan anti-L523S antibody, and detecting the binding between L523S proteinand the antibody in the malignant cells, but not in the normal orreactive cells; thereby producing cells having L523S protein bound tothe anti-L523S antibody, wherein the malignant cells are melanoma cells.2. The method of claim 1, further comprising: (c) contacting thebiological sample with an antibody to a second protein that is a markerof cell lineage, and detecting the binding of the antibody to the secondprotein; thereby producing cells having L523S protein bound to theanti-L523S antibody and the second protein bound to the antibody to thesecond protein.
 3. The method of claim 2, wherein the protein is amarker of cell lineage.
 4. The method of claim 2, wherein the secondprotein is calretinin.
 5. The method of claim 1, further comprising: (d)contacting the biological sample with an antibody to a third proteinwhose expression is restricted to normal or reactive cells, anddetecting the binding of the antibody to the third protein; therebyproducing cells having L523S protein bound to the anti-L523S antibody,the second protein bound to the antibody to the second protein, and thethird protein bound to the antibody to the third protein.
 6. The methodof claim 5, wherein the third protein is desmin.
 7. The method of claim1, wherein at least one detecting step comprises detecting animmunohistochemical stain or flow cytometric evaluation.
 8. The methodof claim 1, wherein the antibody is a monoclonal antibody.
 9. The methodof claim 1, wherein the biological sample is a tissue biopsy, biologicalfluid, cell monolayer, or cell pellet derived from a biological fluid.10. The method according to claim 9, wherein the tissue biopsy isobtained from a pigmented skin lesion.
 11. The method according to claim9, wherein the biological fluid is lung pleural effusion.
 12. The methodof claim 1, wherein step (b) further comprises: exposing the biologicalsample to a labeled secondary antibody that reacts with the antibodybound to L523S protein; and detecting the presence of the secondaryantibody bound to the antibody bound to L523S protein.
 13. The method ofclaim 1, wherein step (b) further comprises: exposing the biologicalsample to a labeled reagent comprising an enzyme-conjugated polymerbackbone that carries secondary antibody molecules that react with theantibody bound to L523S protein; and detecting the presence of thelabeled reagent.
 14. The method of claim 5, wherein the biologicalsample is a tissue biopsy, and wherein step (a) is performed on a firstportion of the tissue biopsy, step (b) is performed on a second portionof the tissue biopsy, and step (c) is performed on a third portion ofthe tissue biopsy.
 15. The method of claim 14, wherein the first andsecond portions are different microtomed sections from the same tissuebiopsy.
 16. The method of claim 14, wherein the first, second and thirdportions are different microtomed sections from the same tissue biopsy.17. The method of claim 2, wherein step (c) further comprises: exposingthe biological sample to a labeled secondary antibody that reacts withthe antibody bound to the second protein; and detecting the presence ofthe secondary antibody bound to the antibody bound to the secondprotein.
 18. The method of claim 2, wherein step (c) further comprises:exposing the biological sample to a labeled reagent comprising anenzyme-conjugated polymer backbone that carries secondary antibodymolecules that react with the antibody bound to the second protein; anddetecting the presence of the labeled reagent.
 19. The method of claim5, wherein step (d) further comprises: exposing the biological sample toa labeled secondary antibody that reacts with the antibody bound to thethird protein; and detecting the presence of the secondary antibodybound to the antibody bound to the third protein.
 20. The method ofclaim 5, wherein step (d) further comprises: exposing the biologicalsample to a labeled reagent comprising an enzyme-conjugated polymerbackbone that carries secondary antibody molecules that react with theantibody bound to the third protein; and detecting the presence of thelabeled reagent.